Sensors for determining parameters in physiological samples are widely used in various fields of chemistry, biology and physiology. The presence and concentration of substances which are degradable by bioactive molecules, such as enzymes, may be determined using sensors comprising a suitable enzyme or enzymes. Such sensors are often referred to as biosensors. Biosensors employing both electrochemical and photometric principles are known.
The determination of creatinine in samples of physiological fluids, such as whole blood, serum or urine, is important for assessing renal function. Creatine phosphate is stored in the muscles of vertebrates and provides an energy reserve. It is irreversibly converted into creatinine (a degradation product) and the energy rich phosphate group. During normal muscle function about 1-2% per day of the total amount of creatine phosphate is converted into creatinine. Creatinine is released into the blood and removed by the kidneys. In a healthy individual the level of creatinine is thus relatively constant at about 35 to about 75 μM. If the level of creatinine in the blood increases it may be a sign of some malfunction of the kidneys. In such cases the level of creatinine may increase to levels as high as 2,000 μM.
Creatinine may be measured by biosensors comprising various enzymes, such as creatinine iminohydrolase (by detection of NH3) or creatinine amidohydrolase. Creatinine amidohydrolase is also referred to as “creatininase”.
In case of creatininase, the creatinine level in a physiological fluid is determined by a cascade of enzymatic reactions resulting in the formation of H2O2, which in turn may be detected amperometrically or photometrically. In some systems a further enzyme (e.g., peroxidase) or an indicator may be used (e.g., a luminophor).
The cascade of enzymatic reactions involving the enzymes creatininase (EC 3.5.2.10), creatine amidinohydrolase (EC 3.5.3.3—“creatinase”) and sarcosine oxidase (EC 1.5.3.1) is represented by the following reactions:

The intermediate product creatine is also present in samples of blood, serum or urine as such. Therefore, a dual sensor system is preferably employed if the creatinine is to be determined by the above cascade of enzymatic reactions. Using a dual sensor system the creatininase may be determined as the difference between the total of the two substances and the intermediate product alone. Accordingly, in a first sensor for the determination of the total concentration of creatinine and creatine in the sample, both creatininase, creatinase and sarcosine oxidase are present for converting creatinine and creatine into H2O2. In a second sensor for the determination of the concentration of creatine in the sample, creatinase and sarcosine oxidase are present for converting creatine into H2O2. The concentration of creatinine in the sample is thus determined from the difference between the total concentration of creatinine and creatine in the sample and the concentration of creatine in the sample.
Such a sensor system is disclosed in US published application 2004/0072277, which is derived from WO 02/14533 (Roche Diagnostics), published Feb. 21, 2002, wherein the H2O2 produced is detected amperometrically. The possible presence of enzyme inhibitors is discussed, particularly the inhibition of creatinase. A stable response of the dual sensor system is obtained for at least one week when measuring a sample of bovine serum containing about 90 μM creatinine.
The enzyme creatininase has been characterised in Kaoru Rikitake et al., “Creatinine Amidohydrolase (Creatininase) from Pseudomonas putida,” J. Biochem. 1979, 86(4), pp. 1109-1117. Creatininase is a metalloenzyme carrying two zinc atoms in each subunit. It appears that the purified enzyme is very stable, especially after heat treatment, which is part of the purification process. Divalent metal ions of Mn, Co, Mg, Zn, Ni, Ca and Fe are shown to inhibit slightly the activity of the purified, heat-treated enzyme. In addition, creatininase shows only slight inhibition by ethylenediamine tetraacetic acid (EDTA) indicating that the zinc atoms are held firmly by the enzyme. This observation is also confirmed by the fact that the zinc ions may only be removed prior to the heat treatment of the enzyme and not from the heat-treated enzyme preparation. Upon removal of the zinc ions from creatininase by specific treatment of a cell digest, the resulting inactive apoenzyme is shown to be reactivated by addition of 0.5 mM of the divalent metal ions of Mn, Co, Mg, Zn, Ni or Fe. Similarly, Inouye et al., “Purification and Characterization of Creatinine Amidohydrolase of Alcaligenes Origin”, Chem. Pharm. Bull. 34(1) 269-274 (1986), disclose that metal ion depleated creatininase can be reactivated by addition of a 1.0 mM MnCl2 solution.
This characterisation of creatininase is confirmed in Tadashi Yoshimoto et al., “Crystal Structures of Creatininase Reveal the Substrate Binding Site and Provide an Insight into the Catalytic Mechanism,”J. Mol. Biol. 2004, 337, pp. 399-416.
Thus, the enzyme creatininase is known to be a stable enzyme only slightly responsive to EDTA and other inhibitors.
Moreover, EP 0 872 728 A1 discloses a biosensor having a reaction layer comprising an enzyme and an electron acceptor, and having in the vicinity thereof a divalent water-soluble salt. The salts are preferably calcium salts, cadmium salts, manganese salts, magnesium salts or strontium salts; preferably chlorides, nitrates or sulfates. The minimum essential concentration of the metal for causing the metal to bind to the enzyme is described as 0.01 mg/mL corresponding to a minimum essential molar concentration of around 180 μM for manganese.
It has been found that when a dual sensor system of the above type is used for multiple measurements of creatinine in physiological samples, the response of the creatinine sensor starts to decrease after a certain period of time or a certain number of measurements. A gradual decrease in the sensitivity of the dual sensor system is the result. This decrease in sensitivity is a disadvantage, since it results in a short use life of the sensor system. Such sensors contain creatininase which is heat-treated, and as such, are expected to be insensitive to metal ion activation.
The relatively high concentrations of manganese apparently required for reactivating of metal-depleated enzyme (Rikitake et al. and Inouye et al.) or incorporation in a biosensor (EP 0 872 728 A1) will often be considered problematic, in particular when used in a multi-sensor apparatus. High concentrations in cleaning or rinse solutions may cause the precipitation of manganese salts, in particular if such solutions mix up with other solutions having different salt concentrations or pH values or with hypochlorite solutions. Moreover, the divalent manganese ion is an electroactive species which may cause unstable zero-currents in electrochemical sensors. Also, manganese may cause interference with magnesium sensors.